the roles of pol and env in the assembly pathway of human foamy...

JOURNAL OF VIROLOGY,0022-538X/98/$04.0010

May 1998, p. 3658–3665 Vol. 72, No. 5

Copyright © 1998, American Society for Microbiology

The Roles of Pol and Env in the Assembly Pathwayof Human Foamy Virus

DAVID N. BALDWIN AND MAXINE L. LINIAL*

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104,and Department of Microbiology, University of Washington, Seattle, Washington 98195

Received 8 August 1997/Accepted 3 February 1998

Human foamy virus (HFV) is the prototype of the Spumavirus genus of retroviruses. These viruses have agenomic organization close to that of other complex retroviruses but have similarities to hepadnaviruses suchas human hepatitis B virus (HBV). Both HFV and HBV express their Pol protein independently of theirstructural proteins. Retroviruses and hepadnaviruses differ in their requirements for particle assembly andgenome packaging. Assembly of retroviral particles containing RNA genomes requires only the Gag structuralprotein. The Pol protein is not required for capsid assembly, and the Env surface glycoprotein is not requiredfor release of virions from the cell. In contrast, assembly of extracellular HBV particles containing DNArequires core structural protein and polymerase (P protein) for assembly of nucleocapsids and requires surfaceglycoproteins for release from the cell. We investigated the requirements for synthesis of extracellular HFVparticles by constructing mutants with either the pol or env gene deleted. We found that the Pol protein isdispensable for production of extracellular particles containing viral nucleic acid. In the absence of Env,intracellular particles are synthesized but few or no extracellular particles could be detected. Thus, foamy virusassembly is distinct from that of other reverse transcriptase-encoding mammalian viruses.

Human foamy virus (HFV) is the prototype of the Spuma-virus genus of the Retroviridae family (35). It was originallyisolated in 1971 from tissue culture cells derived from a humancancer patient (1), but, based on homology with other primateisolates, it is now thought to be of chimpanzee origin (19, 41).HFV is a complex retrovirus which contains several accessorygenes in addition to the canonical retroviral gag, pol, and envgenes (13). One of these genes, bel1 or tas, encodes a transac-tivator protein (22, 36) which is required for viral transcriptionand replication (29). HFV Pol is a 127-kDa polyprotein con-sisting of protease (PR), reverse transcriptase (RT), RNase H(RN), and integrase domains (IN) (31). Unlike other retrovi-ruses, however, HFV expresses Pol independently of Gag froma spliced subgenomic RNA (11, 27, 47). Expression of Pol as aGag-Pol fusion protein, in the case of other retroviruses, pro-vides mechanisms for both expression of Pol and assembly ofthe viral enzymes into particles through Gag-Gag interactions(18, 21). In addition, for most retroviruses, Pol is completelydispensable for virus assembly and RNA packaging, with bothprocesses depending entirely upon Gag (32, 42). While the Envprotein determines tissue tropism for the mature virion, it isdispensable for assembly, packaging, and budding (42).

Pararetroviruses have evolved very different mechanisms forpackaging viral RNA and for budding. Hepadnaviruses such ashuman hepatitis B virus (HBV) require their RT (P protein)for encapsidation of genomic RNA (3, 33, 34). Assembly isinitiated by binding of the nascent P protein to a secondarystructure in the viral RNA called epsilon (ε), after which capsidprotein dimers are recruited to complete capsid formation (5).Capsids form in the cytoplasm and are thought to be trans-ported to the rough endoplasmic reticulum, where they acquirethe surface glycoproteins during budding into the lumen of theendoplasmic reticulum (ER). In contrast to retroviruses, HBV

requires expression of the surface glycoproteins for release ofextracellular particles (7).

While HFV shares features with both retroviruses and theother mammalian RT encoding-viruses such as hepadnavi-ruses, its replication pathway is distinct from both of these.As in the case of HBV, cell-free virions contain significantamounts of DNA (47, 49), indicating either that reverse tran-scription is initiated prior to assembly or that the Pol protein ishighly active inside virions. However, unlike HBV, where amajority of virions contain nicked, circular double-strandedDNA (30), only about 10% of HFV particles contain full-length double-stranded DNA (47). While both HBV and HFVexpress their Pol proteins independently of their structuralproteins, they initiate reverse transcription by different mech-anisms. In HBV, reverse transcription is coupled to assemblywhich is initiated by Pol binding to HBV RNA (4, 34, 45). HFVRNA contains a primer binding site for tRNALys, and all evi-dence suggests that reverse transcription proceeds by a retro-viral pathway (23). The roles of Gag, Pol, and Env in HFVassembly are not known. One recent study suggested that ex-pression of Gag alone did not lead to particle formation (12).

In this study, we have examined the roles of Pol and Env inthe assembly of HFV. We demonstrate that as with otherretroviruses, the Pol protein is not required for particle assem-bly or encapsidation of genomic RNA. In contrast, we havefound that like hepadnaviruses, the HFV Env protein is re-quired for production of extracellular virions but not for intra-cellular particle formation.

MATERIALS AND METHODS

Recombinant plasmid DNAs. HFV proviral deletion mutants DPol569 andDEnv190 were generated by linearizing the pHSRV13 (35) plasmid at uniquePacI and BspEII restriction sites, respectively, digestion with Bal31 exonuclease,and religation after treatment with Klenow DNA polymerase. DPol569 andDEnv190 contain 569- and 190-bp deletions, respectively, which both result intranslational frameshifting. The env mutant DMN (deleted from MroI [position6957] to NdeI [position 8970]), was provided by Martin Lochelt.

The vector pSGC11 was used to make probes for RNase protection analysis ofHFV nucleic acids. pSGC11 contains a 427-bp fragment of the HFV long ter-minal repeat which was amplified by PCR from the pHSRV13 proviral plasmid

* Corresponding author. Mailing address: Division of Basic Sci-ences, Fred Hutchinson Cancer Research Center, A3-015, 1100 Fair-view Ave. N., P.O. Box 19024, Seattle, WA 98109-1024. Phone: (206)667-4442. Fax: (206) 667-5939. E-mail: [email protected].

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with primers containing BamHI and EcoRI restriction sites. These were used forcloning into pBluescript SKII1 (Stratagene). The oligonucleotide primers wereU3BAMP11 (59-ACTTGGATCCGATAATGTTTTAAGGAATACT-39) andU5ECOP11 (59-AGCTGAATTCTGTATATTGATTATCCTAAGG-39).

Cells and culture. FAB indicator cells (foamy virus activation of b-galactosi-dase), described elsewhere (48), were maintained in Dulbecco’s modified Eagle’smedium (DME) supplemented with 5% fetal bovine serum (FBS) and 0.4 mg ofhygromycin per ml. Before transfection, the cells were trypsinized and placed inDME with 5% FBS only. Human embryonic lung fibroblasts (HEL) were main-tained in DME with 10% FBS.

Transient transfection. FAB cells were transfected with proviral constructs byusing Lipofectamine reagent (Gibco-BRL). Liposome complexes were generatedby diluting 5 mg of plasmid DNA in 750 ml of DME lacking penicillin andstreptomycin (P/S2) and adding 750 ml of DME P/S2 containing 25 ml ofLipofectamine reagent. This mixture was allowed to stand for 30 min while theFAB cells were rinsed with DME P/S2. The mixture was added to a 10-cm platewith cells at 75% confluency (approximately 106 cells per plate) and rockedgently before the addition of another 6 ml of DME P/S2. Transfection mixtureswere incubated for 2 to 3 h at 37°C under 6% CO2 and washed twice with isotonicbuffer, and 8 to 10 ml of DME with P/S and 5% FBS were added to each plate.

Expression and purification of recombinant proteins. The central domain ofHFV Gag and the RNase H domain of Pol were cloned into pGM484 foroverexpression in Escherichia coli. Nucleotide sequences 1494 to 2658 and 4635to 5978 of HSRV13 were amplified by PCR with primers containing 59 BamHIor 39 EcoRI restriction sites and three in-frame CACCAT (six-His tag) repeatsat the 59 end of the target sequence. Protein expression was induced in E. coliJDBE3 by addition of 1 mM IPTG (isopropyl-b-D-thiogalactopyranoside) duringlog-phase growth for 4 h. Both recombinant proteins were recovered from theinsoluble fraction of the cells after lysis by sonication. Denatured proteins werepartially purified by Ni1 column chromatography as recommended by the col-umn manufacturer (Qiagen) and further purified by denaturing polyacrylamidegel electrophoresis.

Antibodies. New Zealand White rabbits were immunized with recombinantproteins corresponding to the central region of the HFV Gag protein and theRNase H domain of the Pol protein for the generation of polyclonal monospe-cific antisera (see the previous section). Pure proteins were isolated by polyacryl-amide gel electrophoresis and homogenized in Freund’s incomplete adjuvent forinjection. Antiserum against this domain of Gag recognizes the 78-kDa Gagprecursor and the 74-kDa cleavage product. Antiserum against the RNase Hdomain of Pol recognizes the 127-kDa Pol precursor and the 80-kDa productfrom which integrase has been cleaved by the viral protease.

RIPA. Transiently transfected FAB cells were washed with DME 24 h post-transfection, and labeled for 12 h with [35S]methionine (NEN Research Prod-ucts) at 50 mCi/ml in DME lacking Met and Cys and containing 5% dialyzedFBS. The supernatants were passed through 0.45-mm Nalgene syringe filters, andthe virus was pelleted through 20% sucrose cushions. Virus pellets were resus-pended directly in antibody buffer (20 mM Tris [pH 7.5], 50 mM NaCl, 0.5%Nonidet P-40 [NP-40], 0.5% sodium dodecyl sulfate [SDS], 0.5% deoxycholate[DOC], 0.5% aprotinin, ex tempora 10 mM iodacetamide) for the radioimmu-noprecipitation assay (RIPA). Plates (10-cm) of cells were disrupted in 1 ml ofAb buffer, and chromosomal DNA was sheared by passing the extract through a23-gauge needle. Cell debris were pelleted by microcentrifugation, and incorpo-ration was measured by trichloroacetic acid precipitation. Lysates were incubatedfor 2 h at room temperature in the presence of protein A-Sepharose (Pharmacia)and 2 ml of rabbit antiserum (either anti-Gag or anti-Pol). Protein A beads werewashed twice with high-stringency RIPA buffer (10 mM Tris [pH 7.4], 150 mMNaCl, 1% NP-40, 1% DOC, 0.1% SDS, 0.5% aprotinin), once with high-saltbuffer (10 mM Tris [pH 7.4], 2M NaCl, 1% NP-40, 1% DOC), and then once withTris-EDTA (TE). The beads were boiled for 5 min in 23 denaturing SDS-polyacrylamide gel electrophoresis Laemmli sample buffer, and samples wereelectrophoresed through 10% acrylamide gels. Quantitation was performed witha PhosphorImager and ImageQuant software.

RNase protection assay (RPA). Total nucleic acids from concentrated virionsor whole cells were detected with a Direct Protect kit (Ambion). Purified nucleicacids were treated with RNase-free DNase or DNase-free RNase and thendiluted in lysis buffer for further analysis. Radiolabeled RNA probe was gener-ated with pSGC11 (see above) for in vitro transcription with T7 RNA polymeraseand [32P]UTP (Dupont; 3,000 Ci/mmol) after digestion with EagI. Quantitationwas performed with a PhosphorImager and ImageQuant software.

Intracellular capsid analysis. Transiently transfected FAB cells were washedwith isotonic buffer 36 h posttransfection and lysed by two different methods.Half the plates were lysed gently with Triton X lysis buffer (0.25 M sucrose, 0.5%Triton X-100, 10 mM Tris [pH 7.5], 1 mM EDTA, 140 mM NaCl), and the otherhalf were lysed completely in RIPA Ab buffer to solubilize all intracellularstructures. Lysates were cleared of cellular debris by centrifugation at 2,000 rpmin an IEC HN-SII clinical centrifuge for 30 min and then placed on top of a 20%sucrose cushion and centrifuged at 24,000 rpm in an SW28 rotor (Beckman) for3 h. Pellets were analyzed for the presence of intracellular particles by Westernblotting (immunoblotting) with Gag antiserum. The RIPA Ab buffer-generatedpellets were treated with DNase before SDS-polyacrylamide gel electrophoresis.Samples were electrophoresed through 10% acrylamide gels and transferred toImmobilon-P (polyvinylidene difluoride) membranes with an Ellard Instrumen-

tation Inc. semi-dry transfer apparatus at 0.8 mA/cm2 for 1 h. Transfer bufferconsisted of 15% methanol, 192 mM glycine, and 25 mM Tris-base. The mem-branes were blocked for 1 h with phosphate-buffered saline–5% nonfat dry milkand then probed with Gag antiserum at 1:2,000 in block–0.05% Tween for 2 h.The membranes were washed three times with block–Tween and the probed withdonkey anti-rabbit immunoglobulin Ig conjugated with horseradish peroxidase(Amersham) at 1:7,000 in block1Tween for 2 h. After the membranes werewashed in block-Tween and then straight phosphate-buffered saline for a total of30 min, Gag/donkey anti-rabbit immunoglobulin-conjugated horseradish perox-idase-specific bands were detected by enhanced chemiluminescence as recom-mended by the manufacturer (Amersham).

Electron microscopy. Thin-section transmission electron microscopy (TEM)was performed on FAB cells, which were transiently transfected with DPol569,DEnv190, and wild-type HFV (HSRV13). At 36 h posttransfection, the cells werefixed in Karnofsky’s reagent for sectioning and further staining. The cells wereharvested 36 h posttransfection and prepared for sectioning and staining aspreviously described (2). The sections were analyzed under a JEOL 100SXtransmission electron microscope at 80 kV.

RESULTS

The Env glycoprotein is required for efficient budding fromthe cell but not for particle formation. We were interested inthe role of the Env glycoprotein in the HFV assembly pathway.A deletion mutant (DEnv190) was used to analyze virus assem-bly and RNA packaging. We predicted that such a mutantwould assemble and bud normally from the cell, since it waswild type for Gag and Pol proteins and therefore would con-tain RNA as is the case of murine leukemia virus and otherretroviruses. Transient expression was used to compare theassembly of virus particles and RNA packaging in the presenceor absence of Env. Transfections were performed in FAB cellswhich contain an HFV LTR driving b-galactosidase expression(48); thus, only cells containing the Bel 1 (Tas) transactivatorprotein will turn blue after being stained with the chromogenicsubstrate X-Gal (5-bromo-4-chloro-3-indolyl-5-b-D-galactopy-ranoside). This allowed comparison of transfection efficiencyprior to virus particle and protein analysis. Equivalent trans-fection efficiencies were observed with wild-type and mutantproviruses, usually in the range of 10 to 20%.

To investigate intra- and extracellular viral protein expres-sion, RIPA was performed with Gag antiserum. The majorproducts detected with this serum are of 74 and 78 kDa. Unlikeother retroviruses, no cleavage to mature Gag proteins is de-tected (17). Intracellular expression levels of Gag (Fig. 1A,lanes 1 and 3) were similar for wild-type and DEnv190 asmeasured by RIPA. However, no detectable Gag protein waspresent in the supernatant of DEnv190-transfected cells (lane8), indicating that extracellular virus was not produced in theabsence of the Env glycoprotein. Quantitative analysis of thewild-type bands corresponding to extracellular Gag indicated a20-fold difference between wild-type and DEnv190 at 36 hposttransfection (lanes 6 and 8), which was the limit of thesensitivity of the assay. We also looked at Pol protein expres-sion. The major proteins detected by our RNase H antiserumare a 127-kDa precursor and an 80-kDa Pro-Pol (PR-RT-RN)product from which the 40-kDa integrase domain has beencleaved (31). We found no difference in intracellular Polexpression between HFV wild type (Fig. 1B, lane 1) andDEnv190 (lane 3). Since no particles were detected in thesupernatant, it was not surprising that no RNA could be de-tected (Fig. 2B, lane 5). A different env mutant, DMN (pro-vided by Martin Lochelt, Heidelberg, Germany), was alsotested for comparison. This mutant, whose deletion spans amuch larger portion of the env gene, yielded the same result(data not shown). The DMN construct had also been previ-ously characterized for correct expression of the other majorHFV genes (28) and does not interfere with the essentialinternal promoter for Bel 1 transcription (28). At very late

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times after transfection of these mutants, a very small amountof viral Gag could be detected in the culture supernatant (datanot shown). However, some of this signal could be Gag re-leased from dying cells.

We next examined the effect of the Env deletion on intra-cellular particle formation. The strategy was to lyse wild-type-or DEnv190-transfected cells in different detergent-containingbuffers. Lysis buffer containing Triton-X should lyse cell mem-branes but not disrupt intracellular capsids, whereas lysis withSDS should disrupt the integrity of virions as well as mem-branes. Methods similar to this have been used to quantitateintracellular retroviral type D capsids (Mason-Pfizer monkeyvirus) as well as HBV nucleocapsids (26, 37). Figure 3A indi-cates the amount of viral Gag present in pellets after ultracen-trifugation of both Triton X and SDS lysates. We found similarlevels of pelletable Gag from Triton X lysates for both wild-type-transfected (lane 1) and DEnv190-transfected (Lane 2)cells. No Gag was recovered in pellets after complete lysis inSDS-containing Ab buffer (lanes 4 and 5) or from mock-trans-fected cells (lane 3). Conversely, little or no Gag protein wasdetectable in the ultracentrifugation supernatants of TritonX-lysed cells (Fig. 3B, lanes 1 and 2), whereas wild-type andDEnv190 Gag were detected at similar levels in the SDS lysissupernatants, in which capsid structures are disrupted (lanes 4and 5). These results indicate that the intracellular Gag seen inDEnv190-transfected cells, like wild-type-transfected cells, ispresent in assembled particles and not as free protein. Thus,we conclude that Env is not required for the assembly ofintracellular capsids but is required for the release of virionsfrom the cell.

TEM was performed to verify that particles assembled in theabsence of Env resemble the wild type. We were able to detectintracellular particles in FAB cells transiently transfected withboth wild-type and DEnv190 DNA. DEnv190 particles wereseen in intracellular compartments (Fig. 4D); they resemblewild-type particles (Fig. 4A) and were occasionally found bud-ding from intracellular membranes (Fig. 4E). No particles weredetected at or near the cell surface, and none were found

budding from the plasma membrane. Preformed cytoplasmiccapsids could also be found in both wild-type-transfected (Fig.4B) and DEnv190-transfected (Fig. 4F) cells.

The Pol protein is not required for RNA packaging. Unlikeother retroviruses, HFV Pol is expressed independently ofGag. We were interested to find whether the HFV assemblypathway is dependent on the Pol protein, as is the case forHBV. A pol deletion mutant (DPol569) was used to evaluatewhether the Pol protein was required for RNA packaging. Theviral protease, encoded within the Pol precursor, cleaves the78-kDa Gag precursor near the C terminus to release 74- and4-kDa products. We used RIPA to analyze Gag and Pol pro-tein expression in HFV wild-type- and DPol569-transfectedcells. As expected, we did not see any cleavage of intracellularGag (Fig. 1A, lane 2) or virion-associated Gag from superna-tants (lane 7) of DPol569-transfected cells. Also as expected,no Pol protein could be detected in DPol569-transfected cells(Fig. 1B, lane 2).

Viral RNA was quantitated by RPA. The probe (pSGC11)used in these experiments distinguishes RNA from DNA, sinceit spans part of U3, all of R, and part of U5 (Fig. 2A). DNA(either endogenous viral DNA or plasmid from the transfec-tion) protects an RNA probe fragment corresponding to 427nucleotides (nt). Genomic viral RNA protects two fragmentsof the riboprobe: the 59 end of the genome protects a 330-ntfragment of the probe corresponding to R-U5, and the 39 endof the genome protects a 260-nt fragment corresponding toU3-R. Under the conditions used for hybridization in this as-say, RNA-RNA hybrids are detected more readily than RNA-DNA hybrids (data not shown). The signal in lanes 3 and 4 ofFig. 2B is from RNA, as determined by comparison to themolecular weight markers, although virion DNA is easily de-tected in virus isolated from the media of acutely infected cells(data not shown). Virus used for RPA assays was isolated assoon as it was detectable by RIPA with anti-Gag antiserum inthe supernatants (36 h posttransfection) in an effort to com-pare assembly prior to spread of wild-type virus. We found thatsimilar levels of RNA were detected in wild-type HFV and

FIG. 1. RIPA of cellular and viral Gag and Pol proteins. Assay conditions and antibodies are described in Materials and Methods. (A) RIPA with anti-Gagantiserum. Lanes 1 to 4 contain cellular Gag proteins from transfected cells. Lanes: 1, wild-type (wt) HFV; 2, DPol569; 3, DEnv190; 4, mock-transfected FAB cells. Lane5 contains virus from the supernatant of labelled, acutely infected HEL cells. Lanes 6 to 9 contain extracellular viral Gag proteins from the supernatants of thetransfections. Lanes: 6, wild-type HFV; 7, DPol569; 8, DEnv190; 9, mock-transfected cells. Lanes 6 and 7 were used to normalize the number of wild-type HFV andDPol569 particles for the packaging experiments. (B) RIPA with anti-Pol antiserum. Cellular lysates for this figure were identical to those used with the anti-Gagantiserum. Lanes: 1, wild-type HFV; 2, DPol569; 3, DEnv190; 4, mock-transfected cells.

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DPol569 particles (Fig. 2B, lanes 3 and 4). To verify the findingthat genomic RNA is packaged by the Pol mutant, nucleicacids were purified from virus particles and subjected to treat-ment with RNase-free DNase (lanes 11 and 16) or DNase-freeRNase (lanes 10 and 15) prior to RPA. Both wild-type andDPol569 signals disappeared after treatment with RNase butnot DNase. This demonstrates that the probe is discriminatingbetween RNA and DNA in the RPA reactions and that RNAis packaged by the DPol569 mutant.

As a control in these studies, we compared the DPol569mutant to a protease point mutant (D/A) since it was shownthat it assembles particles and expresses full-length Pol proteinbut is not replication competent (24). We considered that if thePol protein was required for packaging, this mutant wouldserve as a positive control in the absence of viral spread. We

found that the assembly, RNA packaging, and budding of D/Aparticles were identical to those for DPol569 (data not shown).

To quantitate the amount of RNA packaged per virion,particles were normalized to viral Gag protein by RIPA (Fig.1A, lanes 6 and 7). PhosphorImager analysis was used to de-termine the efficiency of RNA packaging for DPol569 relativeto the wild type. Ratios of RNA (Fig. 2B, lanes 3 and 4) to Gag(Fig. 1A, lanes 6 and 7) indicate that the DPol569 mutantpackages genomic RNA with wild-type efficiency. This exper-iment was repeated four times, each time determining theGag/RNA ratio for both wild-type HFV and DPol569 and thencalculating the efficiency of packaging by comparing wild-typeHFV(Gag/RNA) and DPol569(Gag/RNA) values. The averagepackaging efficiency of wild-type HFV with respect to DPol569was 0.96 with a standard deviation of 60.31.

FIG. 2. RPA of wild-type and mutant HFV nucleic acids. (A) The pSGC11 RNA probe is depicted, along with the predicted fragments protected by viral nucleicacids. The open arrow depicts the antisense probe, and the solid arrow represents the direction of viral transcription. (B) Results of RPA. Lanes 2, 7, 12, and 17 containpBR322 MspI-digested, radiolabelled marker DNA. Other lanes: 1, 1% of total undigested free probe; 3, untreated wild-type (wt) HFV; 4, untreated DPol569; 5,untreated DEnv190; 6, mock-transfected cell supernatant; 8, untreated wild-type HFV; 9, mock-digested wild-type HFV; 10, RNase-treated wild-type HFV; 11,DNase-treated wild-type HFV; 13, untreated DPol569; 14, mock-digested DPol569; 15, RNase-treated DPol569; 16, DNase-treated DPol569. Lanes 3 to 6 were usedfor quantitation.



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The Pol protein is not required for virus assembly or bud-ding. TEM was used to analyze particle formation in the ab-sence of the Pol protein. Pol mutant particles were detected byTEM in both intracellular compartments as well as at theplasma membrane (Fig. 4C). The intracellular (left) and extra-cellular (right) particles formed by the DPol569 mutant (Fig.4C) are very similar to those of the wild type (Fig. 4A). The polmutant particles are detected in the extracellular media atwild-type levels, indicating that budding is not impaired by thelack of Pol protein. This data is consistent with results obtainedwith a protease active-site mutant of HFV (D/A) and indicatesthat cleavage or partial cleavage of the Gag protein is notrequired for virus assembly or budding. These results demon-strate that virus assembly occurs in the absence of the Polprotein, as in other retroviruses.

DISCUSSION

While assembly of conventional retroviruses occurs withouteither the Pol protein or the Env protein, we demonstrate herethat HFV assembly is unique. Transient expression of proviralHFV pol and env mutants in FAB cells has permitted us toevaluate the roles of Pol and Env during the late stages of HFVreplication. We have found that like all other retrovirusesstudied, HFV Pol is not required for assembly, RNA packag-ing, or budding. Particles produced by a mutant which is lack-ing the Pol protein resemble wild-type HFV (Fig. 4). Thisfinding confirms previous data that the activity of the viralprotease is not required for particle formation or release fromthe cell. However, it is in contrast to a previous report in whichit was suggested that a protease point mutant (D/A) assemblesaberrantly and that cleavage of the Gag precursor is requiredfor normal virion morphology (24). Although it is possible that

our mutant (DPol569), which retains a portion of the proteaseopen reading frame, has some protease activity, no Gag cleav-age could be detected with anti-Gag antiserum (Fig. 1A, lanes2 and 7). DPol569 packages wild-type levels of RNA as mea-sured by RNase protection analysis of viral nucleic acids iso-lated from extracellular particles.

Analysis of the env deletion mutant gave an unexpectedresult. Although Gag and Pol expression from the env mutantwas equivalent to that from the wild type, extracellular parti-cles were undetectable by RIPA (Fig. 1A, lane 8) or RNaseprotection (Fig. 2B, lane 5). Intracellular particles were, how-ever, detected by TEM. Preformed capsids were found in thecytoplasm (Fig. 4F), some budding was seen (Fig. 4E), andsome particles appeared within membrane-bound cytoplasmicstructures such as the lumen of the rough endoplasmic retic-ulum, the Golgi complex, or secretory vesicles (Fig. 4D). Com-parison of intracellular particle levels demonstrated that Env isnot required for wild-type levels of capsid assembly and thatthis function is mediated by Gag alone (Fig. 3). While the roleof Gag in assembly seems to be retrovirus-like, intracellularparticle retention in the absence of Env is atypical for mostretroviruses, which do not require the Env protein for buddingor particle release from the cell (42). However, this finding isreminiscent of the hepadnavirus assembly pathway in whichthere is a strict requirement of surface glycoproteins for par-ticle release but not nucleocapsid formation (7).

One possible explanation for this observation is that a signalin the Env protein interacts with the secretory machinery andactively induces the transport of enveloped virions to the cellsurface. In the absence of Env, virions could be retained incytoplasmic secretory vesicles. Most cells use a constitutive se-cretory pathway for the transport of membrane componentsand glycoproteins of the extracellular matrix. More specializedsecretory cells can use an additional pathway, which is regu-lated. These cells are able to store soluble proteins and othersubstances in secretory vesicles for regulated release (reviewedin reference 20). If this were the case, we might have expect-ed to see a greater accumulation of env mutant virions with-in intracellular compartments. Also, this type of mechanismwould be highly cell type specific and could have implicationsfor both cytopathicity and virus production. As yet, we have notanalyzed our mutants in cells other than fibroblasts.

Additionally, as with other retroviruses such as human im-munodeficiency virus type 1 (HIV-1), there may be a definableinteraction between the cytoplasmic tail of the Env protein andthe N terminus of Gag (8–10, 14). It may be that this interac-tion enhances Gag multimerization and hence particle assem-bly. If the interaction between Gag and Env is required forefficient particle formation, it is also possible that expression,modification, and localization of mature Env protein are therate-limiting steps in mature-particle formation. In HIV-2, thecytoplasmic tail of the Env glycoprotein is important in en-hancing particle release (6, 39). However, there was only abouta 10-fold effect, comparable to the enhancement of particlerelease by the Vpu protein of HIV-1 (43, 44). The foamy virusenv mutant phenotype is therefore more similar to that of HBVenvelope protein mutants, which are not released in the ab-sence of their surface glycoproteins (7).

Multimerization of Gag is likely to be concentration depen-dent (46), and if interaction with Env at a specific membraneoccurs, it could enhance particle formation at that membrane.It has been demonstrated that the matrix (MA) domain of Gagcan target the structural polyproteins to specific locations inthe cell and direct the site of assembly. In one case, a singlepoint mutation in MA changed a type D retrovirus to type C,which was able to bud from the plasma membrane without

FIG. 3. Comparison of intracellular particle formation by wild-type andDEnv190 by using Western blot analysis. Cell lysates were prepared as discussedin Materials and Methods. (A) Pellets. Lanes: 1 to 3, Triton X-lysed cells; 4 and5, SDS-lysed cells. Lanes 1 and 4 contain wild-type HFV; lanes 2 and 5 containDEnv190; lane 3 contains mock-transfected cells. (B) Supernatants. Lanes: 1 to3, Triton X-lysed cells; 4 to 6, SDS-lysed cells. Lanes 1 and 4 contain wild-typeHPV; lanes 2 and 5 contain DEnv190; lanes 3 and 6 contain mock-transfectedcells.



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FIG. 4. TEM analysis of wild-type and mutant HFV particles. (A and B) Representative examples of wild-type HFV-transfected cells. (C) DPol569-transfected cells.(D to F) DEnv190-transfected cells. For wild-type (A) and DPol569 (C) enveloped particles, intracellular virions are shown on the left and extracellular virions are shownon the right. Cytoplasmic capsids are shown only for wild-type (B) and DEnv190 (F) capsids. Descriptions for fixing and staining of transfected FAB cells can be foundin Materials and Methods.

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preforming capsids (38). Recently it has been reported thatnonbudding, immature intracellular A-type particles can be di-rected to the plasma membrane for assembly and release byaltering the endoplasmic reticulum targeting signal within Gag(46). Although nuclear localization of HFV Gag has been re-ported, it does not seem to be required for replication (40),and no other targeted localization has been described.

In addition, it has been observed that both HFV and HBVsurface glycoproteins contain ER retrieval signals (16, 25)while other retroviral Env proteins do not. The biological rel-evance of this observation for HFV remains to be determined,although it has been reported that the HFV ER retrieval signalis not required for intracellular budding or budding from theplasma membrane. Mutants with mutations in the ER retrievalsignal bud more often from the plasma membrane but releasefewer particles with than does the wild type (15). These dataare consistent with the idea that colocalization of Gag and Envmay occur more efficiently at the membranes of the ER, en-hanced by the ER retrieval signal in Env, but they do notexplain why particles which still form intracellularly withoutEnv are not released from the cell.

There appears to be an evolutionary relationship betweenfoamy viruses and other retroviruses, as well as hepadnavirusessuch as HBV. Several features of HFV replication are of par-ticular interest in this regard. Like HBV, HFV expresses RTindependently of the structural proteins. HFV lacks extensiveproteolytic processing of the Gag structural protein (17) andcontains GR-box nucleic acid binding motifs instead of thecanonical retroviral Cys-His boxes (13). In addition, infectiousdouble-stranded DNA can be extracted from a small percent-age of extracellular HFV virions (49). However, the HBV as-sembly pathway is initiated and completed very differently fromthat of retroviruses. The P protein (RT and RNase H) initiatesthe assembly process and is required for genome encapsidation(3, 34), and surface glycoproteins are required for the releaseof virus from the cell (7). Thus, while HFV may represent anevolutionary bridge between retroviruses and hepadnaviruses,its replication is distinct from both.

ACKNOWLEDGMENTS

D.N.B. was supported by Public Health Service National ResearchService Award T32 GM07270, from the National Institute of GeneralMedical Sciences. This investigation was also supported by grantCA18282 from the National Cancer Institute to M.L.L.

We thank Michael Emerman for critical comments on and review ofthe manuscript, Martin Lochelt for stimulating discussion regardingthis research during his sabbatical at FHCRC, and Scott Eastman forassistance with rabbit antisera.

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